Herpes Simplex Virus 1 (HSV-1) affects millions of individuals, and is the single largest cause of infectious blindness in the U.S. HSV-1 establishes life-long latency in sensory neurons, where abundant viral gene expression is limited to the latency associated transcript (LAT). HSV-1 remains latent as a circular episome associated with histones in sensory neurons, but has the ability to reactivate in response to stressors. This reactivation causes recurrent outbreaks, which is responsible for the majority of HSV-1-related disease and the associated health care costs. Unfortunately, the molecular mechanisms that regulate latency and HSV-1 reactivation are not well defined. A major focus of our research is to determine the molecular mechanisms that control HSV-1 latency and contribute to recurrent HSV-1 infections. Recently, we found that epigenetic marks present on the HSV-1 genome change as the virus reactivates from latency. We have also identified clusters of DNA binding motifs for the cellular insulating protein, CTCF around genomic regions of HSV-1 that are critical for both the establishment of latency as well as in reactivation. These binding motifs are enriched in CTCF during latency and we have already characterized three CTCF sites as chromatin insulators. The overall hypothesis to be tested in this proposal is that the cellular insulating protein, CTCF, regulates HSV-1 gene expression during latency through long-range chromatin interactions that can be destabilized to facilitate HSV-1 reactivation. In Aim 1, we will characterize the insulator function of the CTCF binding motifs in HSV-1 using chromatin immunoprecipitation assays, and map the histone profiles around these CTCF binding motifs. This will allow us to characterize additional elements within the HSV-1 genome that might be responsible for facilitating HSV-1 gene expression during latency. We will also use chromatin conformation capture (3-C) assays to determine whether CTCF regulates HSV-1 gene expression through classic insulator function (in a linear manner) or indirectly through the formation of chromatin loop domains. Finally, we will determine whether mutations to the CTCF binding domains in HSV-1 affect HSV-1 gene expression during latency. In Aim 2 of this proposal, we will be investigating the role of CTCF-mediated gene expression during in vivo reactivation using the highly efficient rabbit ocular model to characterize ocular pathogenesis and CTCF-mediated HSV-1 gene expression during reactivation in vivo. The proposed studies will provide key insight into how the HSV-1 genome is regulated epigenetically, as well as define the role of CTCF and chromatin insulators in HSV-1 gene regulation during latency and in reactivation. In addition, this work will likely increase our understanding of how CTCF regulates cellular transcription from chromosomal elements. .